Protein Phosphate in the Regulation of Protein Phosphorylation and Insulin Secretion

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dc.contributor.advisorRobert Easom
dc.contributor.committeeMemberPorunelloor Mathew
dc.contributor.committeeMemberMing-Chi Wu
dc.creatorParameswara, Vinay K.
dc.date.accessioned2019-08-22T21:15:28Z
dc.date.available2019-08-22T21:15:28Z
dc.date.issued2003-05-01
dc.date.submitted2013-09-23T13:42:07-07:00
dc.description.abstractParameswara, Vinay K., Protein Phosphatase 2A in the Regulation of Ca2+- Sensitive Protein Phosphorylation and Insulin Secretion. Doctor of Philosophy (Biomedical Sciences), May 2003; 191 pp., 28 illustrations; 5 tables; 250 references. Type 2 diabetes is characterized by insufficient insulin secretion in the midst of increased demand from concomitant insulin resistance of peripheral tissues. More specifically, the diabetic β-cell is characterized by impaired responsiveness to D-glucose, the primary physiological regulator of insulin secretion, necessitating that the mechanism of glucose-induced insulin secretion from the β-cell of the pancreas is critically dependent on an elevation of cytosolic calcium as a trigger signal but is also dependent on reversible protein phosphorylation. Accordingly, a number of protein kinases are activated by glucose, or by incretin hormones that enhance glucose-induced insulin secretion. This dissertation however stems from a general hypothesis that protein phosphorylation and insulin secretion may also be controlled via the regulation of protein phosphatases (PP). Initially, a panel of specific antibodies was used to profile the expression of known PP species in the β-cell. By immunoblotting cultured clonal β-cells, INS-1, were shown to express various protein phosphatases namely PP 1, 2A, 2B, 2C, 4 and 6, but with distinct subcellular localization suggesting that these phosphatases regulated distinct functions within the β-cells. Of particular interest, PP-2A holoenzyme was localized to purified fractions of insulin secretory granules suggesting an involvement in insulin regulation. Selective inhibition of PP-2A in the presence of endothall or low concentrations of okadaic acid, increased insulin secretion in the presence of glucose in INS-1 cells. In order to discern potential substrates of PP-2A and thus-mechanisms of action, microcystin immobilized to sepharose was employed to affinity purify phosphatase species from β-cell lysates and proteins complexed with them. Fractions containing PP-2A also contained synapsin I and a specific interaction of these proteins was confirmed by co-immunoprecipitation from INS-1 cell lysates. In contrast, PP-1 was not associated with synapsin I. That synapsin I is indeed a substrate for PP-2A in INS-1 cells was confirmed via the demonstration that synapsin I phosphorylation was increased by okadaic acid under conditions that increased insulin release. Okadaic acid also induced the autophosphrylation and activation of CaMKII, a Ca2+-dependent kinase that phosphorylates synapsin I; suggesting CaMKII may mediate PP-2A effects on insulin secretion. The elimination of syanpsin I, markedly modulates glucose homeostasis of mice and subtly modulates insulin release. In summary these studies document that the modulation of PP-2A in β-cells dramatically influences insulin secretion reinforcing a concept that the control of protein phosphatase may have a critical role in the regulation of insulin secretion. These data suggest that a role of PP-2A on insulin secretion is mediated in part through the regulation of CaMKII activity and synapsin I-phosphorylation.
dc.format.mimetypeapplication/pdf
dc.identifier.urihttps://hdl.handle.net/20.500.12503/29194
dc.language.isoen
dc.provenance.legacyDownloads0
dc.subjectCell and Developmental Biology
dc.subjectCell Biology
dc.subjectCells
dc.subjectCellular and Molecular Physiology
dc.subjectChemicals and Drugs
dc.subjectDiseases
dc.subjectLife Sciences
dc.subjectMedical Cell Biology
dc.subjectMedicine and Health Sciences
dc.subjectNutritional and Metabolic Diseases
dc.subjectProtein phosphate 2A
dc.subjectregulation
dc.subjectCa2+
dc.subjectProtein phosphorylation
dc.subjectinsulin secretion
dc.subjecttype 2 Diabetes
dc.subjectglucose
dc.subjectβ cell
dc.subjectsynapsin I
dc.subjectokadaic acid
dc.subjectCaMKII
dc.titleProtein Phosphate in the Regulation of Protein Phosphorylation and Insulin Secretion
dc.typeDissertation
dc.type.materialtext
thesis.degree.departmentGraduate School of Biomedical Sciences
thesis.degree.disciplineBiomedical Sciences
thesis.degree.grantorUniversity of North Texas Health Science Center at Fort Worth
thesis.degree.nameDoctor of Philosophy

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